Drug discovery programs for oncology typically select compounds which have a predilection for inducing cytotoxic effects in cancer cell lines versus non-cancer cells and, subsequently, for inhibiting the growth of the transplanted cancer cells in the flanks of immuno-compromised mice.
While there has been significant progress over the last decades and a number of new cancer drugs is available today, drugs for the treatment of cancer generally suffer from several problems.
One problem is that drugs for the treatment of cancer often have significant side-effects, including, for example, hair loss, problems with the fingernails and toenails, skin irritation, nausea, vomiting, fatigue, impairment of memory, concentration problems, diarrhea or constipation, anemia, swelling of limbs, lymphedema, weakening of the immune system which may result in infections, bone loss and osteoporosis, impairment of fertility, sexual side effects, incontinence, or second cancers caused by cancer treatment.
Another problem is that, despite cytotoxic effects in vitro and inhibition of tumor growth in vivo additional complications may arise because of the existence of a small subtype of cells called cancer stem cells (CSCs). Such cells are relatively resistant to therapy and are able to, after treatment with the cytotoxic drug has ended, effect repopulation with cancer cells in vivo.
The cancer stem cell hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of the tumor (McDermott & Wicha). There are now numerous studies which have identified cancer stem cells in leukemia, breast cancer, brain cancer, lung cancer, colon cancer, and others (Frank et al., 2010).
To cause relapse, CSCs must have survived primary treatment. A number of factors may underlie this phenomenon, including stem cell quiescence, protected niche environment, upregulated expression of xenobiotic efflux pumps, and enhanced anti-apoptotic and DNA repair pathways.
The first identification of breast cancer stem cells was defined by the combined expression of cell surface markers CD44+/CD24−/low/lin−. As few as 200 of these cells generated tumors in NOD/SCID mice whereas 20,000 cells that did not display this phenotype failed to generate tumor (Al-Hajj et al.).
Later studies suggested that aldehyde dehydrogenase 1 (ALDH-1), a detoxifying enzyme responsible for oxidation of retinol to retinoic acid, may be an even more potent marker of breast CSCs (Ginestier et al.; Morimoto et al.; Charafe-Jauffret et al.). ALDH-1-positive breast CSCs can induce tumor formation with as few as 500 cells. Breast cancer cells that expressed ALDH-1 were more likely to be estrogen receptor (ER) negative, progesterone receptor (PR) negative, and human-epidermal growth factor receptor type 2 (HER-2) positive, and frequently developed distant metastases. ALDH-1-positive cells are resistant to conventional chemotherapy with paclitaxel and epirubicin (Tanei et al.).
Previous studies have shown that adult stem cells can be identified by a side population (SP) phenotype. A SP isolated from the breast cancer cell line MCF7 was found to represent a small percentage of the total cell line and it contained the tumorigenic fraction, as demonstrated by transplantation experiments in NOD/SCID mice xenografts. This fraction was also able to reconstitute the initial heterogeneity of the cell line (Kondo et al.; Patrawala et al.).
In breast tumors, the use of neoadjuvant regimens showed that conventional chemotherapy could lead to enrichment in CSCs in treated patients as well as in xenografted mice (Li et al.; Yu et al.).
This suggests that many cancer therapies, while killing the bulk of tumor cells, may ultimately fail because they do not eliminate CSCs, which survive to regenerate new tumors.
This can be seen, e.g., from the example of breast cancer, the most common cancer in American women, and the second most leading cause of death from cancer in US women despite early detection. Approximately 30% of all patients treated for early-stage disease ultimately develop recurrence, mostly metastatic. For patients with metastatic disease at diagnosis, conventional chemotherapies are initially effective in disease control, but ultimately most patients relapse over time. Recent advances in technology have demonstrated the existence of breast cancer stem cells. These cells are currently believed to be responsible for treatment failures because of their resistance to conventional treatment.
Thus, there remains an urgent need for new pharmaceutical compounds and compositions that have less side effects and that allow to (also) effectively eradicate and target cancer stem cells. There is a need in the art for improved ways to treat cancer, such as breast cancer and leukemia. In particular, there is a need in the art for ways of treatment that have lower side effects. Moreover, there is a need in the art for improved ways to treat cancer in ways that attack a cancer at its primary site (i.e. the site of its origin) and simultaneously reduce/prevent metastasis of the cancer from its primary site to other sites. Moreover, there is a need in the art for improved ways to treat cancer in ways that specifically target cancer stem cells (such as sphere forming cells, colony forming cells, ALDH positive cells, Side Population cells, or CD44HIGH/CD24LOW cells). Moreover, there is a need in the art for new ways to manipulate cultured cells, in particular new ways to block progression of the cell cycle and/or induce apoptosis in cultured cells.
These objects are solved by the below-described aspects of the present invention and the preferable embodiments described.
In a first aspect, the present invention relates to a benzo-thiazolo-imidazole compound having the structure represented by Formula I:
                wherein                    R1, R2, R4 and R5 are independently selected from the group consisting of                            hydrogen,                halogen (preferably F, Cl, Br, or I),                hydroxyl,                amino which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                nitro,                cyano,                thiol,                sulfonyl,                carbonyl, preferably an aldehyde, ketone, ester or amide,                carboxyl,                straight or branched alkyl, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                straight or branched alkoxy, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                straight or branched alkenyl, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                substituted or unsubstituted cycloalkyl, and                substituted or unsubstituted aryl or heteroaryl, preferably substituted or unsubstituted phenyl,                                    wherein, preferably, each of R1, R2, R4 and R5 comprises up to 18, preferably up to 14, more preferably up to 10, more preferably up to 7, more preferably up to 4, more preferably up to 3, more preferably up to 2, more preferably up to 1 carbon atoms,            wherein, more preferably, each of R1, R2, R4 and R5 is hydrogen;            R3 is selected from the group consisting of                            hydrogen,                halogen (preferably F, Cl, Br, or I),                hydroxyl,                amino which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                nitro,                cyano,                thiol,                sulfonyl,                carbonyl, preferably an aldehyde, ketone, ester or amide,                carboxyl,                straight or branched alkyl, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                straight or branched alkoxy, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                straight or branched alkenyl, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                substituted or unsubstituted cycloalkyl, and                substituted or unsubstituted aryl or heteroaryl, preferably substituted or unsubstituted phenyl,                wherein, preferably, R3 comprises up to 18, preferably up to 14, more preferably up to 10, more preferably up to 7, more preferably up to 4, more preferably up to 3, more preferably up to 2, more preferably up to 1 carbon atoms,                                    wherein, more preferably, R3 is methoxy or halogen, more preferably methoxy, F or Cl, more preferably methoxy;            R6 is selected from the group consisting of                            hydrogen,                halogen (preferably F, Cl, Br, or I),                hydroxyl,                amino which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                nitro,                cyano,                thiol,                sulfonyl,                carbonyl, preferably an aldehyde, ketone, ester or amide,                carboxyl,                straight or branched alkyl, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                straight or branched alkoxy, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                straight or branched alkenyl, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                substituted or unsubstituted cycloalkyl, and                substituted or unsubstituted aryl or heteroaryl, preferably substituted or unsubstituted phenyl,                wherein, preferably, R6 comprises up to 18, preferably up to 14, more preferably up to 10, more preferably up to 7, more preferably up to 4, more preferably up to 3, more preferably up to 2, more preferably up to 1 carbon atoms,                                    wherein, more preferably, R6 is hydrogen;            R7, R8, R9 and R10 are independently selected from the group consisting of                            hydrogen,                halogen (preferably F, Cl, Br, or I),                hydroxyl,                amino which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                nitro,                cyano,                thiol,                sulfonyl,                carbonyl, preferably an aldehyde, ketone, ester or amide,                carboxyl,                straight or branched alkyl, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                straight or branched alkoxy, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                straight or branched alkenyl, which is unsubstituted or substituted, preferably with one or more substituents selected from the group consisting of halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl and heteroaryl,                substituted or unsubstituted cycloalkyl, and                substituted or unsubstituted aryl or heteroaryl, preferably substituted or unsubstituted phenyl,                wherein, preferably, each of R7, R8, R9 and R10 comprises up to 18, preferably up to 14, more preferably up to 10, more preferably up to 7, more preferably up to 4, more preferably up to 3, more preferably up to 2, more preferably up to 1 carbon atoms,                                    wherein, more preferably, each of R7, R8, R9 and R10 is hydrogen;or a pharmaceutically acceptable salt thereof.                        
In one embodiment, said benzo-thiazolo-imidazole compound has the structure represented by Formula I:
                wherein                    R1, R2, R4 and R5 are independently selected from the group consisting of                            hydrogen,                halogen (preferably F, Cl, Br, or I),                straight or branched alkyl, which is unsubstituted,                wherein, each of R1, R2, R4 and R5 comprises up to 4, more preferably up to 3, more preferably up to 2, more preferably up to 1 carbon atoms, and wherein, preferably, at least two, more preferably at least three of the four groups R1, R2, R4 and R5 are hydrogen,                                    wherein, more preferably, each of R1, R2, R4 and R5 is hydrogen;            R3 is selected from the group consisting of                            hydrogen,                halogen (preferably F, Cl, Br, or I),                hydroxyl,                straight or branched alkoxy, which is unsubstituted,                wherein, preferably, R3 comprises up to 4, more preferably up to 3, more preferably up to 2, more preferably up to 1 carbon atoms,                                    wherein, more preferably, R3 is methoxy or halogen, more preferably methoxy, F or Cl, more preferably methoxy;            R6 is selected from the group consisting of                            hydrogen,                halogen (preferably F, Cl, Br, or I),                straight or branched alkyl, which is unsubstituted,                wherein, preferably, R6 comprises up to 4, more preferably up to 3, more preferably up to 2, more preferably up to 1 carbon atoms,                                    wherein, more preferably, R6 is hydrogen;            R7, R8, R9 and R10 are independently selected from the group consisting of                            hydrogen,                halogen (preferably F, Cl, Br, or I),                straight or branched alkyl, which is unsubstituted,                wherein, preferably, each of R7, R8, R9 and R10 comprises up to 4, more preferably up to 3, more preferably up to 2, more preferably up to 1 carbon atoms, and                wherein, preferably, at least two, more preferably at least three of the four groups R7, R8, R9 and R10 are hydrogen,                                    wherein, more preferably, each of R7, R8, R9 and R10 is hydrogen;or a pharmaceutically acceptable salt thereof.                        
In one embodiment, said benzo-thiazolo-imidazole compound has the structure represented by Formula II:
or a pharmaceutically acceptable salt thereof.
In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is a compound or pharmaceutically acceptable salt for use as a medicament.
In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is a compound or pharmaceutically acceptable salt for use in the treatment of cancer.
In one embodiment, said cancer comprises or consists of cells that form a solid or non-solid tumor.
In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is a compound or pharmaceutically acceptable salt for use in the treatment of breast cancer, colon cancer or leukemia.
In one embodiment, the cells of said cancer are characterized by a decreased expression level of the protein Numb, compared to non-cancerous cells, preferably non-cancerous cells of the same cell type, more preferably non-cancerous cells of the same cell type from the same subject, as determined by western blotting.
Preferably, said decrease in the expression level of the protein Numb is a decrease by at least 20%, preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%.
In one embodiment, said use involves the administration of said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof to a patient in need thereof. In one embodiment, said patient is a mammal, preferably a human.
In one embodiment, the route of administration of said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is selected from the group consisting of intranasal administration; oral administration; inhalation administration; subcutaneous administration; transdermal administration; intradermal administration; intra-arterial administration with or without occlusion; intracranial administration; intraventricular administration; intravenous administration; buccal administration; intraperitoneal administration; intraocular administration; intramuscular administration; implantation administration; topical administration, intratumor administration and central venous administration.
In one embodiment, said compound or pharmaceutically acceptable salt is formulated for administration by a route selected from the group consisting of intranasal administration; oral administration; inhalation administration; subcutaneous administration; transdermal administration; intradermal administration; intra-arterial administration, with or without occlusion; intracranial administration; intraventricular administration; intravenous administration; buccal administration; intraperitoneal administration; intraocular administration; intramuscular administration; implantation administration; topical administration, intratumor administration and central venous administration.
In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is administered by intravenous injection or by ingestion. In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is formulated for administration by intravenous injection or by ingestion.
In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is administered daily, preferably once every day. In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is administered once every week. In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is administered once every two weeks. In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is administered once every four weeks. In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is administered by bolus administration. In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is administered for at least one week, preferably for at least two weeks, more preferably for at least one month, more preferably for at least two months, more preferably for at least three months.
In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is administered to said patient at a dosage resulting in a dosage of said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof in the range of from 1 to 200 mg/(kg*day), preferably in the range of from 50 to 200 mg/(kg*day).
In one embodiment, simultaneously to said administration of said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof an effective amount of a chemotherapeutic agent selected from the group consisting of paclitaxel, doxyrubicin, vinblastine, vincristine, vinorelbine, topotecan, carboplatin, cisplatin, pemetrexed, irinotecan, gemcitabine, gefitinib, erlotinib, etoposide, fluorouracil, cyclophosphamide, mercaptopurine, fludarabine, ifosfamide, procarbazine and mitoxantrone is administered to said patient.
A listing of chemotherapeutic agents and the dosage to be used can be found in the 2002 Update of Recommendations for the Use of Chemotherapy and Radiotherapy Protectants: Clinical Practice Guidelines of the American Society of Clinical Oncology, J Clin Oncol. 2002 Jun. 15; 20(12):2895-903.
In one embodiment, no chemotherapeutic agent is administered simultaneously to said administration of said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof. In one embodiment, no chemotherapeutic agent selected from the group consisting of paclitaxel, doxyrubicin, vinblastine, vincristine, vinorelbine, topotecan, carboplatin, cisplatin, pemetrexed, irinotecan, gemcitabine, gefitinib, erlotinib, etoposide, fluorouracil, cyclophosphamide, mercaptopurine, fludarabine, ifosfamide, procarbazine and mitoxantrone is administered to said patient simultaneously to said administration of said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof.
In a second aspect, the present invention relates to a pharmaceutical composition comprising a benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof as defined in any of the embodiments above, and at least one pharmaceutically acceptable carrier, diluent and/or excipient.
In one embodiment, said pharmaceutical composition is a pharmaceutical composition for use as a medicament.
In one embodiment, said pharmaceutical composition is a pharmaceutical composition for use in the treatment of cancer.
In one embodiment, said pharmaceutical composition is a pharmaceutical composition for use in the treatment of breast cancer, colon cancer or leukemia.
In one embodiment, said use involves the administration of said pharmaceutical composition to a patient in need thereof.
In one embodiment, said pharmaceutical composition is administered to said patient at a dosage resulting in a dosage of said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof in the range of from 1 to 200 mg/(kg*day), preferably in the range of from 50 to 200 mg/(kg*day).
In such pharmaceutical composition and the embodiments referring to it, said benzo-thiazolo-imidazole compound, said pharmaceutically acceptable salt thereof, said use, said treatment, said cancer, said administration and said patient are preferably as defined in the first aspect of the present invention or any of the embodiments referring to it.
In one embodiment, said pharmaceutically acceptable carrier or excipient comprises an ingredient selected from the group consisting of an alcohol, dimethyl sulfoxide (DMSO), physiological saline, a lipid based formulation, a liposomal formulation, a nanoparticle formulation, a micellar formulation, a water soluble formulation, a biodegradable polymer, an aqueous preparation, a hydrophobic preparation, a lipid based vehicle, and a polymer formulation.
In a third aspect, the present invention relates to the use of a benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof as defined in the first aspect of the present invention or any of the embodiments referring to it for the manufacture of a medicament for the treatment of cancer.
In one embodiment, said use comprises the administration of said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof to a patient in need thereof.
In such use and the embodiment referring to it, said benzo-thiazolo-imidazole compound, said pharmaceutically acceptable salt thereof, said use, said treatment, said cancer, said administration and said patient are preferably as defined in the first aspect of the present invention or any of the embodiments referring to it.
In a fourth aspect, the present invention relates to a method of treatment of cancer, said method comprising the administration of a benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof as defined in the first aspect of the present invention or any of the embodiments referring to it or a pharmaceutical composition as defined in the second aspect of the present invention or any of the embodiments referring to it to a patient in need thereof.
Preferably, said benzo-thiazolo-imidazole compound, said pharmaceutically acceptable salt thereof, said pharmaceutical composition, said treatment, said cancer, said administration and said patient are as defined in the first aspect of the present invention, the second aspect of the present invention, or any of the embodiments referring to them.
In a fifth aspect, the present invention relates to the use of a benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof in a method for the manipulation of cultured cells.
As the skilled person will appreciate, such a method for the manipulation of cultured cells is a method carried out in vitro.
Preferably, said benzo-thiazolo-imidazole compound and said pharmaceutically acceptable salt thereof are as defined in the first aspect of the present invention or any of the embodiments referring to it.
Preferably, said manipulation is the induction of apoptosis and/or the induction of cell cycle arrest, preferably of cell cycle arrest in mitosis, more preferably of cell cycle arrest in early mitosis.
In one embodiment, said benzo-thiazolo-imidazole compound or pharmaceutically acceptable salt thereof is administered to said cultured cells by including it in or adding it to the culture medium used for cultivation of said cultured cells.
In one embodiment, said cultured cells are cells of a cell line.
In one embodiment, said cultured cells are mammalian cells, preferably human cells.
In one embodiment, said cultured cells are cancer cells, preferably breast cancer cells, colon cancer cells or leukemic cells.
In one embodiment, said cultured cells are cells of a cancer cell line, preferably of a breast cancer cell line, colon cancer cell line or leukemia cell line.
In one embodiment, said cultured cells are characterized by a decreased level of expression of the protein Numb compared to non-cancerous cells, preferably non-cancerous cells of the same cell type, more preferably non-cancerous cells of the same cell type from the same subject, as determined by western blotting.
Preferably, said decrease in the level of expression of the protein Numb is a decrease by at least 20%, preferably by at least 30%, more preferably by at least 40%, more preferably by at least 50%.
In one embodiment, said benzo-thiazolo-imidazole compound is applied to the cells at a concentration of 0.2 μM to 20 μM.
In one embodiment, simultaneously to said administration of said benzo-thiazolo-imidazole compound to said cultured cells, an effective amount of another agent inducing cell cycle arrest and/or apoptosis in cultured cells is administered to said cultured cells, preferably by including it in or adding it to the culture medium used for cultivation of said cultured cells.
In one embodiment, no other agent inducing cell cycle arrest and/or apoptosis in cultured cells is administered to said cultured cells simultaneously to said administration of said benzo-thiazolo-imidazole compound.
The term “substituted”, as used herein, is meant to indicate that a hydrogen atom attached to a member atom within a group is replaced by another atom or group, such as replaced by halogen, hydroxyl, amino, nitro, cyano, thiol, sulfonyl, carbonyl, carboxyl, alkyl, alkoxy, alkenyl, cycloalkyl, aryl or heteroaryl; an example of a substituted alkyl is an alkyl substituted with a hydroxyl group, i.e. a hydroxy-alkyl.
The term “alkyl” refers to a monovalent straight or branched chain, saturated aliphatic hydrocarbon radical. Thus, for example, hexyl isomers are alkyls with six carbon atoms, whereas n-, iso-, sec-, and t-butyl are alkyls with four carbon atoms.
The term “alkoxy” means a group having the formula —O-alkyl, in which an alkyl group, as defined above, is attached to the parent molecule via an oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy (—O—CH3 or OMe), ethoxy (—OCH2CH3 or —OEt), t-butoxy (—O—C(CH3)3 or —OtBu) and the like.
The term “alkenyl” refers to a monovalent straight or branched chain aliphatic hydrocarbon radical containing one carbon-carbon double bond. Thus, hexenyl isomers are alkenyls with six carbon atoms, whereas 1-butenyl, 2-butenyl, 3-butenyl and isobutenyl are alkenyls with four carbon atoms.
The term “cycloalkyl” refers to an unsubstituted (or, optionally, a substituted) group that comprises one or more carbocyclic ring, but that does not comprise an aromatic ring. Thus, for example, cyclohexyl is a cycloalkyl with six carbon atoms, and cyclobutyl is a cycloalkyl with four carbon atoms.
The term “halogen” refers to fluorine, chlorine, bromine, or iodine. If the halogen is a substituent, the term refers to the respective radicals.
The term “aryl” refers to an unsubstituted (or, optionally, a substituted) group that comprises one or more carbocyclic rings of which at least one is an aromatic ring. Examples for aryls are, for example, phenyl or naphthyl.
The term “phenyl”, as used herein, is meant to indicate an unsubstituted (or, optionally, a substituted) phenyl group.
The term “heteroaryl”, as used herein, refers to an unsubstituted (or, optionally, a substituted) group that comprises one or more carbocyclic rings of which at least one is a heteroaromatic ring, wherein the heteroaromatic ring contains from 1 to 4 (preferably 1) heteroatoms independently selected from N, O, and S. The term includes, for example, pyridyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thienyl, furanyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isooxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, and thiadiazolyl, benzofuranyl, indolyl, indazolyl, naphthyridinyl, isobenzofuranyl, benzopiperidinyl, benzisoxazolyl, benzoxazolyl, chromenyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, isoindolyl, benzodioxolyl, benzofuranyl, imidazo[1,2-a]pyridinyl, benzotriazolyl, dihydroindolyl, dihydroisoindolyl, indazolyl, indolinyl, isoindolinyl, quinoxalinyl, quinazolinyl, 2,3-dihydrobenzofuranyl, and 2,3-dihydrobenzo-1,4-dioxinyl.
The terms “KSA-101696” and “compound KSA-101696” are used interchangeably and refer to a compound with the structure shown in FIG. 8.
The compounds of formula I in accordance with the present invention may be produced by a method wherein

The compounds of Formula I were prepared by the reaction of halo ketones 1 (X=halogen) with benzimidazoles 2 in the presence of a base such as potassium hydroxide in EtOH/H2O or triethyl amine in EtOH to give sulphides 3. Cyclization of the latter sulphides afforded the corresponding compounds of Formula I.
“Cancer” as used herein, refers to a diseases caused by the uncontrolled, abnormal growth of cells that can spread to adjoining tissues or other parts of the body. Cancer cells can form a solid tumor, in which the cancer cells are massed together, or exist as dispersed cells, as in leukemia.
The terms “cancer cell” and “cancerous cell” are used interchangeably and refer to a cell characterized by uncontrolled, abnormal growth and the ability to invade another tissue or a cell derived from such a cell. Cancer cell includes, for example, a primary cancer cell obtained from a patient with cancer or cell of a cell line derived from such a primary cancer cell. Examples of cancer cells include, but are not limited to, breast cancer cells, cells of a breast cancer cell line, colon cancer cells, cells of a colon cancer cell line, cancer stem cells, and hematological cancer cells such as cells of myelomas, leukemic cells or lymphoma cells.
“Leukemia” as used herein, refers to a disease involving the progressive proliferation of abnormal leukocytes found in hemopoietic tissues, other organs and in the blood, resulting in increased numbers of leukocytes. “Leukemic cells” refers to leukocytes characterized by an increased abnormal proliferation of cells. Leukemic cells may be obtained from a subject diagnosed with leukemia. The term includes, but is not limited to, acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML), and monocytic leukemia.
“Cancer stem cell”, abbreviated “CSC”, refers to a cell that is capable of self-renewal and differentiating into the lineages of cancer cells that comprise a tumor or hematological malignancy. Cancer stem cells are uniquely able to initiate and sustain the disease. Tumors are maintained by a self-renewing cancer stem cell population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of the tumor (McDermott & Wicha). Cancer stem cells have for example been identified in leukemia, breast cancer, brain cancer, lung cancer, colon cancer, and others (Frank et al., 2010) Cancer stem cells include sphere forming cancer cells, colony forming cancer cells, cells defined by the combined expression of cell surface markers CD44+/CD24−/low/lin− (Al-Hajj et al.), cancer cells positive for the marker aldehyde dehydrogenase 1 (ALDH-1) (Ginestier et al.; Morimoto et al.; Charafe-Jauffret et al.), and side population (SP) cells (Kondo et al.; Patrawala et al.). In breast tumors, the use of neoadjuvant regimens showed that conventional chemotherapy could lead to enrichment in CSCs in treated patients as well as in xenografted mice (Li et al.; Yu et al.).
A “sphere forming cancer cell” is a cancer cell with stem cell properties in which that cells can grow and form spheres in serum-free medium in an ultra-low attachment plate.
A “colony forming cancer cell” is a cancer cell with stem cell properties that can grow from a single cell to form a colony. Such cells may be obtained by culturing cells at low density, e.g. 5000 cells per 60 mm dish plate.
A “CD44+/CD24−/low cell” is a cancer cells with stem cell properties in which cells are highly expressing cell surface marker CD44 and weakly expressing cell surface marker CD24. Such cells may be isolated from a cancer cell line or primary tumor with a Flow Cytometric cell sorter.
An “ALDH-1 positive cell” is a cancer cells with stem cell properties which expresses Aldehyde dehydrogenases-1 enzyme. Such cells may be isolated from a cancer cell line or primary tumor sample with a Flow Cytometric cell sorter using the ALDEFLUOR™ kit.
A “side population cell” is a cancer cell with stem cell properties which is able to efflux the fluorescent DNA-binding dye (Vybrant® DyeCycle™). Such cells may be isolated from a cancer cell line or primary tumor with a Flow Cytometric cell sorter.
The term “treatment”, as used herein, refers to the process of providing a subject with a pharmaceutical treatment, e.g., the administration of a drug, such that a disease or disease state is alleviated, reduced, minimized, halted or even healed, and/or such that the chances of a relapse into the disease state are reduced or a relapse into the disease state is even prevented.
“Pharmaceutically acceptable”, as used herein for example in the context of a pharmaceutically acceptable salt of a compound, refers to a substance (or composition) that is non-toxic to the subject to which it is administered and that thus can be used in the formulation of a pharmaceutical product. If the pharmaceutically acceptable substance is part of a pharmaceutical composition, then the term also implies that the pharmaceutically acceptable substance is compatible with the other ingredients of the said pharmaceutical composition.
Examples of pharmaceutically acceptable salts are addition salts which include, without limitation, the non-toxic inorganic and organic acid addition salts such as the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzenesulfonate derived from benzensulfonic acid, the benzoate derived from benzoic acid, the cinnamate derived from cinnamic acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the formate derived from formic acid, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the hydrochloride derived from hydrochloric acid, the hydrobromide derived from hydrobromic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the malonate derived from malonic acid, the mandelate derived from mandelic acid, the methanesulfonate derived from methane sulphonic acid, the naphthalene-2-sulphonate derived from naphtalene-2-sulphonic acid, the nitrate derived from nitric acid, the perchlorate derived from perchloric acid, the phosphate derived from phosphoric acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acid, the tartrate derived from tartaric acid the sulphate derived from sulphuric acid, the tartrate derived from tartaric acid, the toluene-p-sulphonate derived from p-toluene sulphonic acid, the naphthaline-1,5-disulphonate derived from naphthaline-1,5-disulphonic acid and the like. Such salts may be formed by procedures well known and described in the art.
While the compound according to the present invention for use in therapy may be administered in the form of the raw chemical compound, it is preferred to introduce the active ingredient, optionally in the form of a physiologically acceptable salt, in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries. As the skilled person will appreciate, the ingredients of such a pharmaceutical composition must be compatible with the other ingredients of the formulation and not harmful to the recipient thereof.
The term “pharmaceutically acceptable carrier, diluent and/or excipient” refers to a non-toxic, inert, solid, semi-solid, or liquid diluent material or formulation auxiliary of any type. “Pharmaceutically acceptable” in this context is meant to designate that said carrier is compatible with the other ingredients of the pharmaceutical composition and not harmful to the patient that the pharmaceutical composition is administered to. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, water-propylene glycol solutions, or aqueous polyethylene glycol solutions.
The production of medicaments or pharmaceutical compositions containing a benzo-thiazolo-imidazole compound according to the present invention or a pharmaceutically acceptable salt thereof and their application can be performed according to well-known pharmaceutical methods.
A medicament of the invention may be a medicament suitable for oral, rectal, bronchial, nasal, topical, buccal, sub-lingual, transdermal, parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion) administration, or a medicament in a form suitable for administration by inhalation or insufflation, including powders and liquid aerosol administration.
Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co. Easton, Pa.). The term “effective amount”, as used herein, refers to an amount that produces a desired treatment effect in a subject. This amount will vary depending upon a variety of factors, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. A person skilled in the art will be able to determine an effective amount through routine experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy 20th Edition, Gennaro, Ed., Williams & Wilkins Pennsylvania, 2000.
At some instances, the present application indicates that “simultaneously to” administration of a first agent a second agent is administered. This is meant to designate that the second agent is administered (a) at the same time at which said first agent is applied to the patient, (b) at a time that lies between individual administrations of said first agent, if administration of first agent occurs in several individual administrations, or (c) after administration of said first agent, while the administered first agent is still present in the tissue, blood, or digestive system of the patient or on the surface of the skin of the patient.
“Cultured cells”, as used herein, refers to cells growing under conditions of in vitro culture. Thus cultured cells are free tissue cells that are cultivated in a system entirely apart from their normal environment in which the conditions and factors for growth can be varied at will within the boundaries tolerated by the cells.
As used herein, the term “in vitro” means occurring outside of a living organism. The term in vitro can describe processes/conditions occurring within a cell culture system. In contrast to “in vitro”, the term “in vivo” means occurring within a living organism.
When the present application refers to the “manipulation of cultured cells”, as used herein for example in the context of a certain compound being used in a method for the “manipulation of cultured cells”, this refers to a situation where desired changes are brought about in said cultured cells or their molecular components, typically by applying an effective amount of said compound into contact with said cultured cells and allowing the compound to act on said cultured cells for a time span sufficient to bring about such effects. Manipulations of cultured cells include, for example, inducing cell cycle arrest or inducing apoptosis in said cultured cells.
The present inventors have surprisingly found that the benzo-thiazolo-imidazole compounds according to the present invention, such as the compound with the structure represented in FIG. 8 (referred to also as KSA-101696 in this application), have effects in the treatment of cancer, in particular in the treatment of breast cancer, colon cancer and leukemia. The present inventors have moreover found that, surprisingly, the compounds of this group also have effects on cultured cells, and are for example capable of causing cell cycle arrest of cultured cells in early mitosis and the induction of apoptosis.
Moreover, surprisingly KSA-101696 showed even higher effectiveness on cancer cells with aggressive behavior such as chemoresistance and metastasis than on chemosensitive and non-metastasized (non-aggressive) cancer cells.
Without wishing to be bound by theory, it seems possible that one mechanism how KSA-101696 may affect cells may be by increasing the expression of Numb protein, which is the natural blocker of the Notch pathway.
The Numb protein regulates the Notch-, Hedgehog- and TP53-activated pathways, endocytosis (it is involved in cargo internalization and recycling), determination of polarity (it interacts with the PAR complex, and regulates adherens junctions and tight junctions), and ubiquitination (it exploits this mechanism to regulate protein function and stability). This complex biochemical network lies at the heart of Numb's involvement in diverse cellular phenotypes, including cell fate developmental decisions, maintenance of stem cell compartments, regulation of cell polarity and adhesion, and migration and induced differentiation for therapeutics purposes in humans and animals.
Numb functions as an intrinsic cell fate determinant that is asymmetrically localized in neuronal precursor cells where it influences cell fate by antagonizing signaling from the Notch receptor. Decreased Numb has been demonstrated in mammary carcinomas and higher percentage of the tumors with deficient or reduced expression belonged to the triple-negative (ER−/PR−/HER2−) subgroup (ER: estrogen receptor; PR: progesterone receptor; HER2: human epidermal growth factor receptor 2) compared to tumors with retained Numb expression. Furthermore, decreased expression was associated with poorer distant disease-free survival.
Notch is a fundamental signaling pathway that regulates embryonic cell fate specification. Its driven tumorigenesis in human breast cancer has been suggested by the development of adenocarcinomas in the murine mammary gland following pathway activation and the loss of Numb expression, in a large proportion of breast carcinomas. Furthermore, during pathway activation, Notch intracellular domain (NICD) translocates to the nucleus and binds the CSL (“CBF-1, Suppressor of Hairless, Lag-2”) transcription factor. The NICD/CSL complex induces expression of target genes, including those of the hairy/enhancer of split (Hes) family, the cell cycle regulator p21 and cyclin D1. Cancer stem cells are dependent on a number of key signaling pathways. One of these is the Notch pathway. For example, in breast cancer, it has been reported that the fate of CSCs is controlled by the Notch pathway through induction of Jagged-1. More importantly, self-renewal capacity of mammospheres is enhanced 10-fold when cultured in the presence of a synthetic peptide derived from the DSL (delta-Serrate-Lag2) domain, which is highly conserved in all Notch ligands and capable of Notch receptor activation. On the other hand, the self-renewal capacity was inhibited by Notch 4 blocking antibody or an inhibitor of the γ-secretase enzyme.